The usual method for determining the primary structure of a protein involves proteolytic cleavage, isolation of the peptides, and determination of their amino acid sequences. A major interest in the structure of PLAP is to explain at the molecular level the unusual polymorphism that exists among the phenotypes. Since monoclonal antibodies recognize such differences, we had hoped to bypass the usual procedure of determining the complete protein structure by using monoclonal antibodies to immunoadsorb from proteolytic digests the peptides containing the structural variations. However, the molecular variability identifiable by monoclonal antibodies resides in the large segment of 55 kilodaltons, which cannot be further proteolyzed without denaturation of the molecules as with SDS, reducing agents, and heat. Unfortunately, denaturing conditions that expose other proteolytic sites on the PLAP molecule also destroy the antibody recognition sites on the polypeptide chains, so that further efforts in the approach we were attempting would appear not to be effective. We have used the approaches of proteolysis, antibody binding, and electron microscopy to compile an overall picture of the structure of PLAP and its orientation with respect to the plasma membrane. Further definition of the specificities of the monoclonal antibodies as to which PLAP variants they recognize and studies at the electron-microscopic level visualizing the interaction of PLAP with those antibodies will show whether the variability occurs at only one site or multiple sites on the molecule. Three monoclonal antibodies with distinct antigenic specificities were examined by electron microscopy for their binding to genetic variants of human placental alkaline phosphatase. The variants studied were SS, FS, and FF, which are the most common. In the reaction with the H5 antibody, all three variants of HPLAP preferentially formed circular immune complexes composed of two antibodies and two enzyme molecules. In the reaction with F11 or B2 antibody, the SS variant formed circular complexes, the FS variant formed Y-shaped complexes composed of one antibody and two enzyme molecules, whereas the FF variant scarcely reacted. These results suggest that H5 binds to both S and F subunits, whereas F11 and B2 essentially bind only to the S subunit. Furthermore, the formation of circular complexes in the reaction of the mixture of two antibodies, F11 and B2, with FS variant suggested that these two antibodies bind to different sites on the S subunit. Thus, the F and S subunits appear to differ structurally at more than one site. By localizing the binding sites of F11 and B2, we have postulated that the two subunits of the HPLAP molecule are arranged counter-symmetrically. (M)